The inventors of this application have developed a novel technology for suppressing gene expression termed TRUE (tRNase ZL-utilizing efficacious gene silencing) gene silencing (PTL 1, NPL 1-4). tRNase ZL is a long form of tRNA 3′ processing endoribonuclease (tRNase Z, or 3′ tRNase), which can remove a 3′ trailer from precursor tRNA (NPL 5, 6). TRUE gene silencing is based on an additional unique enzymatic property of mammalian tRNase ZL, which is that it can cleave any target RNA at any desired site by recognizing a pre-tRNA-like or micro-pre-tRNA-like complex formed between the target RNA and artificial small guide nucleic acids (NPL 7-13). Small guide nucleic acid (especially, small guide RNA) is divided into four groups, 5′-half-tRNA (NFL 8), 12-16-nt linear RNA (NPL 13), heptamer-type RNA (NPL 9), and hook-type RNA (NPL 12).
The inventors have demonstrated the efficacy of TRUE gene silencing in various mammalian cells by introducing small guide nucleic acids targeting various mRNAs either as their expression plasmids or as 2′-O-methyl RNAs (NPL 1-4) (hereinafter, the small guide nucleic acid may be referred as “sg nucleic acid”). For example, the HIV-1 expression in Jurkat cells was almost completely suppressed by a 5′-half-tRNA-type sgRNA up to 18 days (NPL 2), and a luciferase gene expression in the mouse livers was inhibited by a heptamer-type sgRNA (NPL 3). Furthermore, it have been shown that the efficacy of TRUE gene silencing can become comparable to that of the RNA interference (RNAi) (NPL 3, 14) and can surpass it in some cases (NPL 4).
Recently, the inventors have found that tRNase ZL exists in the cytosol as well as in the nuclei and that 5′-half-tRNA, which is exactly what they have been using as sg nucleic acid, exists in the cytoplasm (NPL 15). The inventors have also shown that human cytosolic tRNase ZL modulates gene expression by cleaving mRNA under the direction of 5′-half-tRNA and that the PPMIF mRNA is one of the genuine targets of tRNase ZL (NPL 15). Furthermore, the inventors have demonstrated that a subset of human miRNAs including miR-103 can work as hook-type sg nucleic acid and can downregulate gene expression through directing mRNA cleavage by cytosolic tRNase ZL (NPL 16). It is obvious that TRUE gene silencing functions on the basis of the newly unveiled physiological role of cytosolic tRNase ZL directed by cellular small noncoding RNAs.
One of the final destinations of TRUE gene silencing is to generate cancer therapeutic or preventive agents made of sg nucleic acid. The inventors have already shown that the cellular levels of mRNAs encoding Bcl-2 and VEGF, which are promising molecular targets for cancer therapy, can be downregulated under the direction of 5′-half-tRNA-type, 14-nt linear-type, or heptamer-type sg nucleic acid (NPL 1, 4, 17).
From a pharmacological point of view, heptamer-type sg nucleic acid would be the most appropriate for that purpose for the following reasons: heptamers are much easier and cheaper to synthesize than longer sg nucleic acids, and cells can uptake up heptamer-type nucleic acids relatively easily without any stimulating reagents (NPL 18).